Chitosan is an amino-polysaccharide obtained by alkaline deacetylation of chitin, a natural polysaccharide found in the exoskeletons of shellfish and insects. Chitin cannot be dissolved in water except in concentrated mineral acid aqueous solutions, during which dissolution there is a decrease in the degree of polymerization and probably removal of some acetyl groups. Such characteristics have undoubtedly limited its investigation and utilisation in many fields, in spite of the advantages claimed for chitin and its great abundance in nature. In contrast, the numerous industrial applications claimed for chitosan, are in part attributed to its good solubility in mild acidic media, via the formation of ammonium groups.
Conventionally, chitosan is dissolved in aqueous acidic media and can be maintained in solution up to a pH near 6.2 (just below its pKa of ˜6.3). Under these conditions, the reactivity of chitosan is significantly decreased, because of the predominance of non-reactive NH3+ groups compared to NH2 groups, and the latter are known as nucleophilic and therefore susceptible to react with various electrophiles due to their unshared pair of electrons. Nonetheless, a variety of chemical approaches have been employed to homogeneously modify chitosan under acidic conditions (pH<6), specifically by reacting aldehydes, acid chlorides, acid anhydrides and epoxides, and the like, with chitosan's amino groups.
To achieve chitosan modification under homogeneous conditions, prior art reports the addition of an organic co-solvent (methanol, pyridine, etc.) to the acidic chitosan solution, in order to enhance the chitosan reactivity (U.S. Pat. Nos. 4,996,307 and 4,424,346) or the use of a large excess of reagent (Hirano et al., Biopolymers, 15, 1685, 1976, Kubota et al., Polymer Journal, 29, 123, 1997). However, the presence of an organic co-solvent or an excess of reagent is not desired for medical applications. In addition environmental concerns are providing strong incentive for eliminating organic solvent and reducing the use of reactive reagents. Furthermore, at low pH (below 6.2) the number of free amino groups is insufficient to allow the chitosan to undergo a reaction with some electrophilic reagents, particularly those bearing benzoimidate or epoxy groups.
All studies concerned with the N-substitution of chitosan confirm the importance of availability and activation of chitosan's free, non-ionized, amino groups. A recent patent (U.S. Pat. No. 5,977,330) claims the N-substitution of chitosan with good yield via a high activation of chitosan's free amino groups by controlling two factors which enhance the chitosan reactivity, namely, the neutral pH and the use of an organic solvent. However, in addition to an organic solvent, the reaction was heterogeneously performed on re-precipitated chitosan due to the impossibility of maintaining chitosan in solution under neutral pH conditions, when conventional alkali solutions such as NaOH or NH4OH are used as neutralising agents.
It would be highly desirable to be provided with an alternative method to homogeneously modify or cross-link chitosan, by providing an aqueous chitosan solution, which can be maintained quite in solution in the vicinity of neutral pH, since under such conditions the number and the reactivity of free amino groups are considerably enhanced.
It would also be highly desirable to be provided with an alternative method that would allow the elimination of organic solvent and prevent the use of an excess of reagent, and would nonetheless still render possible reactions between chitosan and electrophilic functional groups, which usually require neutral pH to occur.